Journal of Sustainability Science and Management ISSN: 1823-8556 Volume 13 Number 2, December 2018: 15-33 © Penerbit UMT

BIOLOGICAL PROPERTIES AND CHEMICAL DIVERSITY OF flexibilis, AN ALCYONACEAN SOFT CORAL

THILAHGAVANI NAGAPPAN1.2* AND KISHNETH PALANIVELOO3

1 School of Marine and Environmental Sciences, Unversiti Malaysia Terengganu, 21030 Kuala Nerus, Terengganu, Malaysia 2Institute of Marine Biotechnology, Unversiti Malaysia Terengganu, 21030 Kuala Nerus, Terengganu, Malaysia 3Institute of Ocean and Earth Science, Universiti Malaya, Jalan Universiti, 50603 Kuala Lumpur, Malaysia

*Corresponding author: [email protected]

Abstract: Soft corals of the genus Sinularia (order ) are one of the most widespread sessile organisms in the Indo-Pacific waters. Unlike the hard corals, the major portion of soft coral is made up of inorganic skeletons consisting of calcareous spicules surrounded by a thin layer of tissue. This layer of tissue, made up of fleshy colonies with organic matter, is responsible in diversity of secondary metabolites. In order to survive the outburst of algal bloom, dynamic of microbial growth and predation of marine organisms, these soft corals synthesize unique chemicals as part of defense mechanism that makes them unpalatable to most marine life. As such, members of the genus Sinularia exhibits a diversity of secondary metabolites ranging from sesquiterpenes, diterpenes, polyhydroxylated steroids, and polyamine compounds. These metabolites had been shown to possess various biological activities such as antimicrobial, anti-inflammatory and cytotoxicity. This present paper reviews the chemistry and highlights the potential biological activities of metabolites from Sinularia flexibilis and provides a perspective for future research.

Keywords: Sinularia flexibilis, soft coral, secondary metabolites, biological activities. Introduction 2012). Researchers from Australia, India and Japan have isolated and elucidated an extensive The status of marine natural products has been a range of secondary metabolites constituting popular subject for reviews (Kelecom, 2002; sesquiterpenes, diterpenes, polyhydroxylated Proksch et al., 2002; Haefner, 2003; Capon, 2010; steroids and polyamine skeletons. Metabolites Blunt et al., 2015) as this field has resulted in the isolated from the genus Sinularia is reported to development of a substantial number of bioactive display potential bioactivities such as metabolites for the betterment of mankind. antimicrobial, anti-inflammatory and cytotoxic Scheuer (1983) and Faulkner (2002) contributed activities closely related to the high interaction tremendously in creating a platform in the field of in the marine environment (Palaniveloo marine natural product chemistry through & Vairappan, 2014). To date, numerous comprehensive reviews pertaining to chemical publications are currently available pertaining to and biological perspectives of secondary the chemistry and pharmacology of metabolites metabolites. The coral reef represents an isolated from Sinularia flexibilis found in the extraordinary diverse ecosystem in tropical waters of Australia, Japan and India (Kamel et al., environments with soft corals often constituting a 2005; Chen et al., 2010; Anjeneyulu et al., 1997) dominant part of the reef. Sinularia is a genus in as well as extensive research on S. flexibilis from phylum classified in class Alcyonaria, in the Formosan waters (Hu et al., 2013; Duh et al., the family and is widely distributed 1998; Lin et al., 2009; 2013; Lo et al., 2009; from the waters of east Africa to the western 2010) is documented. Over the past 30 years, Pacific, inhabiting the coral reefs or rocks up to more than 15 000 novel secondary metabolites depths of 30 meters (Piccinetti et al., 2017). This have been discovered from this organism (Li et genus is known to consist approximately 90 al., 2006). Since 1995, various research papers species, of which more than 50 have been have been published on investigations of the chemically evaluated (Chen et al., chemical constituents 16 BIOLOGICAL PROPERTIES AND CHEMICAL DIVERSITY OF Sinularia flexibilis, AN ALCYONACEAN SOFT CORAL of the soft coral genus Sinularia reporting new, activities of the chemical constituents from S. novel terpenoids and their pharmacological flexibilis (Fig. 1) which has been a subject of potentials, globally. Hence, this comprehensive interest in the area of marine drug discovery. review focuses on the chemistry and biological

Figure 1: Underwater photographs of S. flexibilis found in the waters of Mantanani Island, Sabah, Malaysia. Terpenoids of Alcyonacean and only sesquiterpenes were isolated from Parerythropodium spp. In the family Investigations involving soft corals started as Nephtheiidae, the genus Nephthea spp comprised early as in 1972 with the isolation of of sesquiterpenes or diterpenes of which 70% prostaglandin [(15R)-PGA2] from Plexaura are cembrane-type skeleton producers. homomalla Esper, a gorgonian whereas the Interestingly, Litophyton spp have only been attention on isolation of secondary metabolites reported to contain cembranoid-type diterpenes from Alcyonacean started in 1970s. Coll (1992) while the genera Lemnalia, Paralemnalia, and reported patterns in chemical distribution across Capnella spp produces sesquiterpenes. Across the various soft coral genera where terpenoids the family Xeniidae, the genera Efflatounaria, have been confirmed as the dominating cluster of Cespitularia and Xenia, Anthelia spp have been secondary metabolites in Alcyonacean. studied with the earlier two genera producing diterpenes, sesquiterpenes and cembrane-type According to Coll (1992), the presence of diterpene. In the latter two genera, 90% of the diterpenes in Sinularia spp and Sarcophyton compounds isolated from Xenia spp and spp is made up of cembrane-type skeleton Anthelia spp belong to the xenicane-type while Lobophytum spp was reported to produce skeleton group. Shown below in Figure 2 (1- a mixture of 70 % cembrane and 30% 19) is the highlight of diverse skeletal pattern of germacrene-type skeleton, respectively. across soft coral genera. The Cladiella spp was found to produce cladiellane-type, cyclized cembrane compounds

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Figure 2: Diversity of isolated chemical skeletal across soft corals genera. Chemical diversity of Sinularia flexibilis al., 2008; Lakshmi et al., 2009; Chen et al., 2010; Hu et al., 2013; Lin et al., 2013; 2009; Genus Sinularia is placed under the soft coral Chen et al., 2012) making them valuable for family Alcyoniidae. In total, 90 species of biomedical research. The secondary Sinularia have been identified worldwide and metabolites isolated from soft corals, apart close to 50 species have been chemically studied from having biomedical potentials, it is also (Chen et al., 2012). Numerous publications serves as chemotaxonomical markers for currently exists pertaining to this genus from Sinularia species identification (Veseveldt, Australian, Japanese and Indian waters (Coll et 1980; Palaniveloo & Vairappan, 2014). al., 1985; Anjeneyulu et al., 1997; Kamel et al., 2005). The secondary metabolites isolated Sesquiterpenes commonly includes sesquiterpenes, diterpenes, polyhydroxylated steroids and polyamine which Among earliest sesquiterpenes discovered from displays a wide array of biological properties such Sinularia was furanosesquiterpenoid acid (20) by as antimicrobial, anti-inflammatory and cytotoxic Coll et al. and co-workers (1977) from Sinularia activities (Anjeneyulu et al., 1997; Duh et al., gonatodes followed by isolation of furanoquinol 1998; Kamel et al., 2005; Khalesi et (21) from Sinularia lochomodes in 1978.

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Furanosesquiterpenoids are commonly known to waters of Okinawa, Japan (Kusumi et al., exhibit a wide range of biological potentials such 1992). This same sesquiterpene skeleton, was as anti-microbial, anti-inflammatory and cytotoxic initially isolated from the soft coral genus activities (Kamel et al.,2005; Chen et al., 2010; Lobophytum by Dunlop and Well (1979) and Yang et al., 2013). However, only one furano- Sinularia dissecta (Reddy et al., 1993; Ramesh type sesquiterpene have been reported from S. et al., 1999; Reddy et al., 2002). Figure 3 flexibilis to date. The compound displays the chemical structures of (+)-β-elemene (22) was isolated along with the furanosesquiterpenoid acid (20), furanoquinol diterpene lobatriene from S. flexibilis from (21) and (+)-β-elemene (22).

Figure 3: The structures of furanosesquiterpenoid acid (20), furanoquinol (21) and (+)-β- elemene (22) from the genus Sinularia. Diterpenes an interesting oxygen moiety bearing hydroxyl group (-OOH) represented by a low field Cembranes are the most common secondary chemical shifts of δ 7.60 – 7.80 ppm. Lo and metabolites isolated from soft corals. This class of co-workers also isolated more cembranoid-type diterpenes possesses a 14-membered ring diterpenes, sinuladiterpenes G (35) - I (37) skeleton. As such, cembranes can be considered a (Fig. 4) as a continuation of their previous work chemotaxonomical marker for soft corals. The documented in 2009 (Lo et al., 2010). earliest of cembrane-type skeleton isolated is pukalide (23), consisting of a butenolide, epoxide, The compound flexibilene (38) is among the isopropenyl and a α,α-disubstituted furan-β- first few cembranes isolated and reported from S. carboxylate moiety derived from Sinularia flexibilis followed by the isolation of sinularin abrupta (Missakian et al., 1975). As for S. (39) and dihydrosinularin (40) reported by flexibilis, sinulariolide (24) was the first diterpene Weinheimer et al. (1977). These compounds were isolated (Tursch et al., 1975). Following the then revised as flexibilide (39) and isolation of sinulariolide (24), its derivatives dihydroflexibilide (40) prior being isolated from comprising of 11-dehydrosinulariolide (25), 11- specimens extracted from Australian waters epi-sinulariolide (26), 11-epi-sinulariolide acetate (Kazlauskas et al., 1978). Next, the Philippines (27) (Fig. 4) was isolated from specimens specimen led to the isolation of sinulariolone collected from Red Sea water (Kashman et al., (41) with a lactone ring. However, sinulariolone 1997) along with 11-epoxysinulariolide (41) as reported to be highly oxygenated, (28) (Mori et al., 1983). These compounds bearing a total of six oxygen atoms (Guerrerro were also isolated from a Taiwanese specimen et al., 1995). of S. flexibilis with additional diterpenes; In addition, the Indian Ocean specimen led to sinuladiterpenes A (29) - F (34) (Lo et al., 2009). Sinuladiterpenes A (29) and B (30) had the isolation of cembranolide-type δ-lactones,

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flexibilolide (42) and dihydroflexibilolide (43) of secondary metabolites from crude extracts of from the ethyl acetate extract (Anjaneyulu et al., the Chinese specimen led to the isolation of 1996). Along with these compounds, cembrene A cembranoid diterpenes, sinuflexolide (47), (44), flexibilene (38) sandensolide (45) as well as dihydrosinuflexolide (48), sinuflexibilin (49) the ϵ-lactone sandensolide monoacetate and the bicembranoid sinuflexin (50) (Duh et (46) (Anjaneyulu et al., 1996; 1997) were also al., 1998) (Fig. 5). discovered. Bioassay-guided isolation

Figure 4: Chemical structures of pukalide (23), sinulariolide (24), its derivatives and Sinuladiterpenes A (29) - I (37).

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Figure 5: Chemical structures of cembrane diterpenes and its derivatives (38-49) isolated from the Indian Ocean S. flexibilis. S. flexibilis have yielded several other sulphated biscembranoid called bicembranoids as well. A study on the Chinese thioflexibilolide A (52). Uniquely, this specimens of this soft coral has led to the compound comprised of structures chemically isolation of a highly oxygenated bicembranoid- identical to sinularin (49) and dihydrosinularin type sinulaflexiolide A (51). In addition to the (40) and is linked with a sulphur atom. Figure 6 isolation of sinuflexin (50) and sinulaflexiolide displays the chemical structures of A (51), Chen and co-workers (Chen et al., bicembranoids isolated from S. flexibilis. 2010) were able to isolate and elucidate a

Figure 6: Bicembranoids sinuflexin (50), sinulaflexiolide A (51) and thioflexibilolide A (52) isolated from Chinese S. flexibilis. In-depth study on the Chinese specimen of S. of 10 more sinulaflexiolides via bioassay-guided flexibilis, had contributed to the collection of techniques were named sinulaflexiolides B (53) - cembranoid-type diterpenes, where the isolation K (62). Most of these sinulaflexiolides are

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structurally, highly oxygenated. The chemical al., 1983) were isolated. Capillolide (66) have structures of sinulaflexiolides B (53) - K (62) been previously reported being isolated from along with its derivatives, acetylsinuflexolide Sinularia capillosa (Su et al., 2000) and Sinularia (63), 11-acetyldihydrosinuflexolide (64), 5- microclavata (Zhang et al., 2008), however this is dehydrosinulariolide (65), capillolide (66), 5,8- the first record of Capillolide (66) being isolated epoxy-9-acetoxysinulariolide (67) and the from S. flexibilis. The structures of these enantiomer of 14-deoxycrassin (68) (Mori et compounds are also shown in Figure 7.

Figure 7: The collection of cembranoid-type diterpenes (53-68) isolated from the Chinese S. flexibilis. Investigation upon Taiwanese S. flexibilis analogue of flexibilisolides and flexibilisin is produced four cembrane-type diterpenes, distingushable by the presence of acetate instead flexibilins A (69), B (76), C (71) and D (72) of hydroxyl moiety in the latter’s structure. As a (Fig. 8) along with sandensolide (Hu et al., continuation of chemical analysis conducted on 2013). This same group of researchers was also this specimen, Shih and co-workers (2012) further successful in isolating flexibilisolides A (73) isolated flexibilisolides C (79) - G (83) (Fig. 8). and B (74), flexibilisins A (75) and B Sinularia flexibilis was also reported to be a (76) together with several other pre-isolated source for flexilarin where a total of 10 compounds. Flexibilisin C (77) was later isolated cembranoid diterpenes flexilarins A (84) - J (93) alongside 11,12-secoflexibilin (78). The (Fig. 9) were isolated.

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Figure 8: Chemical structures of flexibilin (69-72), flexibilisin (75-77) and flexibilisolide (73-83) derivatives from Taiwanese S. flexibilis.

Figure 9: Diversity of flexilarins A (84) - J (93) from the Taiwanese S. flexibilis.

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Apart from cembrane-type diterpenes, Japanese The production of certain metabolite could researchers successfully isolated lobane-type possibly be a natural response of the organism to diterpenes; lobatrienol (94) and lobatriene (95) survive the environmental stress to sustain the from the specimens from Okinawa (Hamada et evolution of species (Coll & Sammarco, 1992). al., 1992). The relative and absolute Apart from this, a cytotoxic cladiellene-type stereochemistry of lobatriene (95) was diterpene named alcyonin (97) was also reported determined via a modified Mosher method from S. flexibilis of the Okinawan waters along (Kusumi et al., 1992). The cultured S. flexibilis with litophynin A (98) and cladiellin (99) from Taiwan was found to produce a quinone (Kusumi et al., 1988). Lithophynin A (98) was derivative, named flexibilisquinone (96), which previously reported from the soft coral, possessed anti-inflammatory properties. Litophyton sp. (Ochi et al., 1987). This was the However, it is still debatable whether the only record of cladiellene-type metabolite from bioactive compound isolated is produced as a this species of soft coral up to 2013. Structures of chemical adaptation in a cultured environment. these compounds are shown in Figure 10.

Figure 10: Chemical skeletons of lobane (94-95), quinine (96) and cladiellene (97-99) type diterpenes. Since 2013, eight additional diterpenes were measurement. Tsai et al. (2015), reported the reported from the S. flexibilis of South China isolation of five new diterpenes from the soft Sea. In 2014, Palaniveloo and Vairappan coral S. flexibilis and Sinularia sandensis, reported the isolation of ten cembrane-type marking a total of six new records for S. diterpene from a single specimen from the flexibilis. The diterpenes diepoxycembrene waters of Kota Kinabalu, Malaysia. However, B (102), dihydromanaarenolide I (103) and all the reported metabolites were known isosinulaflexiolide K (104) were isolated along analogues. In a separate investigation, the S. with 3,4:811-bisepoxysinuacetoxycembra- flexibilis from Malaysia also contained two 15(17)-en-1,12-olide (105), dendronpholide isopropyl (ene)-type cembrene diterpenes. F (106) and dendronpholide G (107). On the These metabolites, (3S,4S,11S,12S,1E,7E)- other hand, (Chen et al., 2015) reported the 3,4:11,12-bisepoxycembra-1,7-diene (100) isolation of eight new metabolites comprising and (1E,3E,7E)-11,12-epoxycembra-1,3,7- of six α-methylene-δ-lactone-bearing triene (101) were isolated and elucidated cembranoids, a 15-membered macrocyclic based on 1D and 2D-NMR spectroscopic diterpenoid, and a biscembranoid. The six

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α-methylene-δ-lactone-bearing cembranoids 15-membered macrocyclic diterpenoid being reported were 9α-hydroxy-flexibilede (108), discovered from a marine source, whereas 15(17)-dehydromanaarenolide (109), 8- sinulaflexiolide L (115) is the third member of dehydroxy- 15(17)-dehydromanaarenolide E the extremely rare cembrane dimers connected (110), 15, 17-dedihydromanaarenolide A (111), through C–C single bond (Chen et al., 2015). 15,17-dedihydromanaarenolide C (112), epi- The chemical structures of new metabolites flexilarin A (113), epoxyflexibilene (114) and reported from S. flexibilis throughout 2014 to sinulaflexiolide L (115). The isolation of the present is highlighted in Figure 11. epoxyflexibilene (114) represents the second

Figure 11: Chemical structures of recent compounds from S. flexibilis.

Sterols 6-en-3β-ol (114), 5α,8α-epidioxygorgosta- 6,9(11)-dien-3β-ol (115), 22α,28- Soft corals synthesize a wide range of epidioxycholesta-5,23(E)-dien-3β-ol (116) and sesquiterpenes and diterpenes that are crucial for 22β,28 -epidioxycholesta-5,23(E)-dien-3β-ol the survival of these organisms in harsh marine (117) (Yu et al., 2006) and ergosterol peroxide environment. However, sterols are also equally (118) isolated from the Chinese S. flexibilis vital for the development of neural system of were also found in marine sponges (Arepalli et these soft-bodied invertebrates. Sterols are al., 2009). This unique group of sterol, 5,8- metabolically active compounds and biosynthesis epidioxy-24-methylcholesta-6,24(28)-dien-3β- of these chemicals is important and could cause ol (119), 5,8-epidioxy-24-methylcholesta- major disorder when a mismatch occurs in the 6,9(11),24(28)-trien-3β-ol (120), 5,8-epidioxy- production and physiological needs (Sarma et al., 24-methylcholesta-6,9(11),22-trien-3β-ol 2009). Among the earliest records, a total of four (121) from the terrestrial mycorrhizal species, sterols, 5α,8α-epidioxygorgosta- Rhizoctonia repens (Gunatilaka et al., 1981)

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and 5,8-epidioxy-24-methylcholesta-6-en-3β-ol 6,9(11),24(28)-trien-3β-ol (120), 5,8-epidioxy-24- (122) and 5,8-epidioxy-23,24-dimethylcholesta-6,22- methylcholesta-6,9(11),22-trien-3β-ol (121) and dien-3β-ol (123) were previously isolated from an 24-methylcholesta-5,24(28)-diene-3,7-diol (126), edible mushroom Grifola frondosa (Yaoita et al., 1998) which were originally reported from the bark of were also found in S. flexibilis. However, 5,8-epidioxy- Amoora yunnanensis were found in the 22,23-methylene-24-methylcholesta-6-en-3β-ol (124) Taiwanese S. flexibilis. Finally, the most recent and 24-methylcholesta-5,24(28)-dien-3β-ol (125) were additions in the list of sterols of S. flexibilis is the reported from an unidentified Sinularia species isolation of two new polyhydroxylated sterols 7a- (Venkateswarlu et al., 1999) and Cladiella species. Luo hydroxy-crassarosterol A (127) and 11-acetoxy- and co-workers (2007), reported the isolation of 5,8- 7a-hydroxy-crassarosterol A (128) (Chen et al., epidioxy-24-methylcholesta- 2015). Figure 12 shows the chemical structures of sterols isolated from S. flexibilis.

Figures 12: Chemical structures of sterols (114-128) isolated from S. flexibilis. Biological properties of Sinularia flexibilis active. Sinulariolide (24) was reported to display antifouling properties and cytotoxicity against Investigation upon biological potentials of cancer cell lines (Tursch et al., 1975; Wang et al., metabolites isolated from this soft coral is still 2017). Its derivative, 11-epi-sinulariolide acetate limited compared to other marine organisms. (27) inhibited iNOS and COX-2 protein of LPS Nevertheless, a number of compounds isolated stimulated on RAW264.7 macrophage from S. flexibilis are reported to be biologically

Journal of Sustainability Science and Management Volume 13 Number 2, December 2018 : 15-33 26 BIOLOGICAL PROPERTIES AND CHEMICAL DIVERSITY OF Sinularia flexibilis, AN ALCYONACEAN SOFT CORAL cells (Lin et al., 2013; Hsu et al., 2013). On the (48) exhibited mild potency against the P-388 other hand, extract of cultured S. flexibilis (murine leukemia) cell line. Sinuflexin (50), a revealed anti-migration, invasion effects and bicembranoid isolated inhibits the P-388 cell isolated 11-epi-sinulariolide acetate (27) line at the IC50 value of 1.32 µg/ml. displays toxicity towards HA22T cells. It was Meanwhile, sinulaflexiolides B (53) - K (62) discovered that 11-epi-sinulariolide acetate displayed inhibitory effects against HL-60 (27) displayed a concentration-dependent (human promyelocytic leukemia cells) at 87%, inhibitory effect on the migration of human HeLa (cervical carcinoma) at 92%, BGC-823 HCC HA22T (hepatocellular carcinoma) cells (human gastric carcinoma) at 93%, Bel-7402 (Wu et al., 2015). This compound suppressed (human hepatocarcinoma) at 52%, PC-3M-IE8 protein levels of matrix metalloproteinase-2 (human prostate carcinoma) at 83% and Hep-2 (MMP-2), matrix metalloproteinase-9 (MMP- (human epithelial type-2 carcinoma) with 83% 9) and urokinase-type plasminogen activator cell inhibition, constantly at a concentration of (uPA) in HA22T cells. Introduction of the 100 µg/ml (Wen et al., 2008; Guo et al., 2016). cembrane-type diterpene also marks tissue The quinone-type diterpene isolated from inhibition of metalloproteinase-1 (TIMP- 1) and metalloproteinase-2 (TIMP-2) in a Taiwanese S. flexibilis displayed anti- concentration-dependent manner, suppression inflammatory properties. Sharing the similar chemical skeleton with diferrent functional of phosphorylation of ERK1/2 and p38MAPK moiety, flexibilisquinone (96) was able to inhibit leading to successful suppression of the the accumulation of iNOS and COX-2 protein of phosphorylation of FAK/PI3K/AKT/mTOR LPS stimulated RAW264.7 macrophage cells (Lin pathways (Lin et al., 2014). et al., 2013). In addition, the cembranoid Sinularin (39) and dihydrosinularin (40) were diterpenes from the same specimen, flexilarin D revised and renamed as flexibilide (39) and (87) displayed an extremely impressive 50% dihydroflexibilide (40) by Weinheimer et al. inhibition of Hep-2 cell line as compared to the (1977). The compounds flexibilide and commercial anti-bladder cancer drug, dihydroflexibilide were reported to display anti- Mitomycin C at 0.07 µg/mL. This compound inflammatory and antiarthritic potential as well as was also found to suppress the proliferation of antimicrobial properties against the Gram-positive HeLa, DAOY (medullablasto carcinoma) and Bacillus subtilis and Staphylococcus aureus MCF-7 (human breast adenocarcinoma) cells (Buckle et al., 1980). On the contrary, 11-epi- by requiring 0.41, 1.24, 1.24 µg/mL to inhibit sinulariolide (26) and its acetate 50% of the cell lines, respectively (Lin et al., (27) only displayed mild inhibition towards 2009). This same group of researchers these bacteria. Meanwhile, Flexibilide (39), evaluated cytotoxicity of flexibilisolides A dihydroflexibilide (40) and sinulariolide (24) (73), B (74), flexibilisins A (75) and B (76), were found to exhibit vascular contractions where all of these compounds exhibited when tested against rat cardiac muscles (Aceret cytotoxicity towards the same set of cell lines et al., 1998). with IC50 values of less than 20 µg/mL, respectively (Dunlop & Well, 1979). The crude extracts of S. flexibilis were also reported to display cytotoxicity towards A549 Flexibilisolide C (79) together with several (human lung adenocarcinoma), HT-29 (human known compounds, 11-dehydrosinulariolide (25), colon adenocarcinoma), KB (human epidermoid 11-epi-sinulariolide (26), 11-epoxysinulariolide carcinoma) and P-388 (mouse lymphocytic (28) and 14-deoxycrassin (68) were reported to leukaemia) cell lines. Bioassay-guided isolation display cytotoxicity of 50% cell inhibition at a of cembranoid-type diterpenes, sinuflexolide (47), concentration of less then 15 µg/mL against dihydrosinuflexolide (48) and sinuflexibilin HeLa and B16 cell lines as well as reduction of (49) (Duh et al., 1998) displayed potential as LPS induced protein, iNOS and COX- 2 in anticancer agents except dihydrosinuflexolide RAW 264.7 macrophage cells (Shih et al.,

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2012) whereas thioflexibilolide A (52) was potential including as immunosuppressive, anti found to reduce LPS stimulated protein, iNOS -inflammatory, antiviral, antiplasmodial and in macrophage cells. Similarly, the compound antitumor properties (Wang et al., 2004). In 3,4:8,11-bisepoxysinuacetoxycembra-15(17)- addition to that, the polyhydroxylated sterol 7a- en-1,12-olide (103) was reported to suppress hydroxy- crassarosterol A (127) exhibited a the production of the iNOS and COX-2 pro- moderate protein tyrosine phosphatase 1B inflammatory cytokines (Tsai et al., 2015). (PTP1B) inhibitory activity with an IC50 value Thioflexibilolide A (52) displayed of 33.05 µM while 11-acetoxy-7a-hydroxy- neuroprotective activity at 0.01 µM with values crassarosterol A (128) displayed weak in vitro of 73.2% suggesting it to have therapeutic cytotoxicities against the tumor cell lines K562 potential for neurodegenerative disease (Chen (myelogenous leukemia) and HL-60 (Chen et et al., 2010). The widely studied sterol from S. al., 2014; Mayer et al., 2003). The summary of flexibilis, ergosterol peroxide (118) is known to compound bioactivity from S. flexibilis is exhibit a range of biological displayed in Table 1.

Table 1: Highlights of bioactivities displayed by metabolites isolated from S. flexibilis globally.

Carcinoma cells Metabolites IC 50 Activity A549 (human lung cell) Crude extracts - HT-29 (human colon cell) Crude extracts - KB (human epidermoid cell) Crude extracts - P388 Crude extracts - (mouse lymphocytic leukaemia) Sinuflexolide - dihydrosinuflexolide - Sinuflexibilin - Sinuflexin 50 % at 1.32 µg/ml HL-60 sinulaflexiolides B - K 87 % at 100 µg/ml (human promyelocytic leukemia) flexilarin D 50 % at 0.41 µg/ml flexibilisolides A - flexibilisolides B - flexibilisins A 50 % at < 20 µg/ml flexibilisins B - HeLa (cervical carcinoma) sinulaflexiolides B – K 92 % at 100 µg/ml Flexibilisolide C - 11-dehydrosinulariolide - 11-epi-sinulariolide 50 % at < 15 µg/ml 11-epoxysinulariolide - 14-deoxycrassin - BGC-823 (human gastric cell) sinulaflexiolides B – K 93 % at 100 µg/ml Bel-7402 (human hepatocarcinoma) sinulaflexiolides B – K 52 % at 100 µg/ml PC-3M-IE8 (human prostate cell) sinulaflexiolides B – K 83 % at 100 µg/ml Hep-2 (human epithelial type-2 cell) sinulaflexiolides B – K 83 % at 100 µg/ml flexilarin D 50 % at 0.07 µg/ml

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Carcinoma cells Metabolites IC 50 Activity flexibilisolides A - flexibilisolides B - flexibilisins A 50 % at < 20 µg/ml flexibilisins B - DAOY (medullablasto cell) flexilarin D 50 % at 1.24 µg/ml flexibilisolides A - flexibilisolides B - flexibilisins A 50 % at < 20 µg/ml flexibilisins B - MCF-7 (human breast cell) flexilarin D 50 % at 1.24 µg/ml flexibilisolides A - flexibilisolides B - flexibilisins A 50 % at < 20 µg/ml flexibilisins B - B16 (melanoma cell) Flexibilisolide C - 11-dehydrosinulariolide - 11-epi-sinulariolide 50 % at < 15 µg/ml 11-epoxysinulariolide - 14-deoxycrassin - RAW264.7 macrophage cells flexibilisquinone - Flexibilisolide C - 11-dehydrosinulariolide iNOS and COX-2 11-epi-sinulariolide - 11-epoxysinulariolide - 14-deoxycrassin - 3,4:8,11-bisepoxysinuacetoxy- - cembra-15(17)-en-1,12-olide thioflexibilolide A iNOS Neuroprotective activity thioflexibilolide A 73.2 % at 0.01 µM PTP1B 7α-hydroxy-crassarosterol A 50 % at 33.05 µM

Conclusion invertebrates. Sea slug, tunicates, sponges are amongst the most active contributors. Hence, this The diversity of chemical analogues and the review paper is aimed to discuss about the discovery of new metabolites from marine Alcyonaria chemistry that has been rapidly organisms are signs that these organisms adapt developed over the past 25 years and is still to survive in extreme environments. Along with progressing. With the diverse chemical scaffolds these adaptations, more diversified metabolites isolated, the biological and pharmacological with a wide array of bioactive potentials are properties associated with these soft corals, produced. As of today, quite a handful of particularly, terpenoids is highly promising and secondary metabolites originating from the excites further investigations. Cembranoid marine environment were obtained from marine diterpenes isolated from S. flexibilis are known

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to exhibit impressive cytotoxic, antitumor and Blunt, J. W., Copp, B. R., Keyzers, R. A., anti-inflammatory activities. In depth Munro, M. H. G., & Prinsep, M. R. (2015). investigation on the differential activity of Marine Natural Products: Review. Natural these metabolites, their mechanisms of action Product Report, 32: 116-211. as well as structure-related activity of marine- derived metabolites are extremely necessary to Buckle, P. J., Baldo, B. A., & Taylor, K. M. develop potent pharmaceuticals for mankind. (1980). The anti-inflammatory activity of More future research in these aspects should be marine natural products 6-n- considered. Recent hybridization and indoor tridecylsalicylic acid, flexibilide and cultivation of soft corals could also contribute dendalone 3-hydroxybutyrate. Agents and to chemical variation as compared to naturally Actions, 10: 361-367. growing S. flexibilis in the marine environment. Capon, R. J. (2010). Marine Natural Products Chemistry: Past, Present, and Future. Acknowledgements Australian Journal of Chemistry, 63: 851- The authors are grateful to the Malaysian Ministry 854. of Science and Technology (MOSTI) and the Chen, B. W., Chao, C. H., Su, J. H., Huang, C. Ministry for Higher Education (MOHE) for the Y., Dai, C. F., Wen, Z. H., & Sheu, J. H. scholarship awards as well as to Universiti (2010). A novel symmetric sulfur- Malaysia Terengganu for supporting this study. containing biscembranoid from the Formosan soft coral Sinularia flexibilis. References Tetrahedron Letters, 51: 5764-5766. Aceret, T. L., Coll, J. C., Uchio, Y., & Sammarco, Chen, B. W., Chao, C. H., Su, J. H., Huang, C. P. W. (1998). Antimicrobial activity of the Y., Dai, C. F., & Wen, Z. H. (2010). A diterpenes flexibilide and sinulariolide novel symmetric sulfur-containing derived from Sinularia flexibilis Quoy and biscembranoid from the Formosan soft Gaimard. Comparative Biochemistry and coral Sinularia flexibilis. Tetrahedron Physiology, 120: 121-126. letters, 51: 5764-5766. Anjeneyulu, A. S. R., Sagar, K. S., & Rao, G. V. Chen, W., Li, Y., & Guo, Y. (2012). Terpenoids (1997). New Cembranoid Lactones from the of Sinularia soft corals: chemistry and Indian Ocean Soft Coral Sinularia flexibilis. bioactivity. Acta Pharmaceutica Sinica B Journal for Natural Products, 60(1): 9-12. (Chinese Traditional Medicine and Natural Product), 2: 227-237. Anjaneyulu, A. S. R., & Sagar, K. S. (1996). Flexibilolide and dihydroflexibilolide, the Chen, W. T., Li-Yan., & Guo, Y. W. (2012). first trihydroxy cembranolide lactones Terpenoids of Sinularia soft corals: from the soft coral Sinularia flexibilis of chemistry and bioactivity. Acta the Indian Ocean. Natural Product Letters, Pharmaceutica Sinica B, 2(3):227-237. 9(2): 127-135. Chen, W. T., Liu, H. L., Yao, L. G., & Guo, Y. Arepalli, S. K., Sridhar, V., Rao, J. V., W. (2014). 9,11-Secosteroids and Kennady, P. K., & Venkateswarlu, Y. polyhydroxylated steroids from two South (2009). Furano-sesquiterpene from soft China Sea soft corals Sarcophyton coral, Sinularia kavarittiensis: induces trocheliophorum and Sinularia flexibilis. apoptosis via the mitochondrial-mediated Steroids, 92: 56-61. caspase-dependent pathway in THP-1, Chen, W. T., Jia, L., Wang, J. R., Li, X. W., & leukemia cell line. Apoptosis, 14:729–40.

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